Gas6 drives Zika virus-induced neurological complications in humans and congenital syndrome in immunocompetent mice
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Evaluation Summary:
The pathophysiology of Zika virus infection remains an area of high research interest. In this study, the authors use diverse experimental models to examine the potential role of Gas6 in Zika virus infection and associated neurological sequelae.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
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Evaluation Summary:
The pathophysiology of Zika virus infection remains an area of high research interest. In this study, the authors use diverse experimental models to examine the potential role of Gas6 in Zika virus infection and associated neurological sequelae.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Silva-Filho et al investigate the role of Gas6 in the pathophysiology of Zika virus (ZIKV) infection, neurologic complications and congenital infection. Starting with human specimens from patients with ZIKV infections, with and without neurologic manifestations, patients with neurologic illnesses unrelated to ZIKV and healthy donors, the authors demonstrate by ELISA that Gas6 is elevated in serum of patients with ZIKV infection, especially those with neurologic manifestations. In patient PBMCs, this finding correlated with an in increase in AXL and SOCS1, both negative regulators of antiviral pathways, and corresponding decrease in IFNβ and IFIT1 mRNA. Ex vivo transcriptional profiling suggested a dose-dependent relationship between Gas6 levels and ZIKV infection.
The authors next use …
Reviewer #1 (Public Review):
In this manuscript, Silva-Filho et al investigate the role of Gas6 in the pathophysiology of Zika virus (ZIKV) infection, neurologic complications and congenital infection. Starting with human specimens from patients with ZIKV infections, with and without neurologic manifestations, patients with neurologic illnesses unrelated to ZIKV and healthy donors, the authors demonstrate by ELISA that Gas6 is elevated in serum of patients with ZIKV infection, especially those with neurologic manifestations. In patient PBMCs, this finding correlated with an in increase in AXL and SOCS1, both negative regulators of antiviral pathways, and corresponding decrease in IFNβ and IFIT1 mRNA. Ex vivo transcriptional profiling suggested a dose-dependent relationship between Gas6 levels and ZIKV infection.
The authors next use warfarin as a model to inhibit Gas6 and show that antiviral responses are restored. Concerns about this approach are that 1) it is indirect, 2) warfarin leads to upregulation of IFNβ and IFIT-1 expression itself and 3) there are no experiments to demonstrate a strict Gas6-dependent effects.
The authors further demonstrate a role for Gas6 in ZIKV pathogenesis in an immunocompetent mouse model. This is a striking finding given that mouse STAT2 is resistant to degradation by flavivirus NS5, and adult IFNAR competent mice are typically refractory to flavivirus infection, which lends support to the hypothesis that Gas6 is mediating a suppression in IFN response. Furthermore, they do demonstrate that Gas6 complexed ZIKV infection in pregnant dams leads to congenital Zika syndrome (CZS) like presentations in offspring.
What is not demonstrated, however, is whether other effects of Gas6, e.g. on vascular proliferation or clotting cascades, could be also mediating these effects. Controlled mouse infections using Gas6 alone and evaluating for manifestations of coagulopathy or hypercoagulability might inform the investigations further. Nonetheless, together, these studies add to our knowledge of the diverse roles of Gas6 in animal pathophysiology.
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Reviewer #2 (Public Review):
Silva-Filho et al. observed that serum Gas6 level is enhanced in ZIKV-infected patients and further enhanced among ZIKV patients with neurological symptoms. Through a range of in vitro and in vivo experiments, the authors concluded that Gas6 levels above a threshold detected in Neuro(ZIKV) patients leads to upregulation of TAM receptors and SOCS-mediated suppression of antiviral immunity, resulting in higher virus loads and pathogenesis.
The authors made many observations: (1) Gas6 levels are increased in ZIKV patients and further increased in ZIKV patients displaying neuropathogenesis, (2) while Gas6 is negatively associated with pro-inflammatory cytokines, this pattern is completely disrupted in Non-Neuro ZIKV patients, although negative correlation is somewhat returned in Neuro ZIKV patients, (3) the …
Reviewer #2 (Public Review):
Silva-Filho et al. observed that serum Gas6 level is enhanced in ZIKV-infected patients and further enhanced among ZIKV patients with neurological symptoms. Through a range of in vitro and in vivo experiments, the authors concluded that Gas6 levels above a threshold detected in Neuro(ZIKV) patients leads to upregulation of TAM receptors and SOCS-mediated suppression of antiviral immunity, resulting in higher virus loads and pathogenesis.
The authors made many observations: (1) Gas6 levels are increased in ZIKV patients and further increased in ZIKV patients displaying neuropathogenesis, (2) while Gas6 is negatively associated with pro-inflammatory cytokines, this pattern is completely disrupted in Non-Neuro ZIKV patients, although negative correlation is somewhat returned in Neuro ZIKV patients, (3) the expression of Axl and Mer receptors, as well as SOCS1, is enhanced in PBMC when infected with ZIKV, (4) viral load in immune-competent mice is increased markedly when inoculating ZIKV was coated with Gas6, (5) subcutaneous infection of immune-competent pregnant mice with Gas6-coated ZIKV resulted in fetal malformation without observable differences in viral loads in the fetus, and (6) intravaginal infection of immune-competent pregnant mice with Gas6-coated ZIKV resulted in a dramatic increase in viral loads without causing fetal malformation.
Several of these observations are novel and important for better understanding ZIKV pathogenesis. However, the manuscript is too complex to provide insights to the field, and some statements are only partially true or unsupported by the data.
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Reviewer #3 (Public Review):
This manuscript investigates the impact of Growth arrest-specific 6 (Gas6) on Zika virus (ZIKV) infection and congenital disease. The authors use a cohort of patient samples isolated from the recent ZIKV outbreak and compliment these data with in vitro and in vivo mouse models to conclude that Gas6 association with ZIKV viral particles promotes neuronal complication and congenital ZIKV syndrome. A clear strength of the study is the incorporation of human data, which allows for some translatability from the in vitro and in vivo mouse studies included in the study. However, despite enthusiasm for the human dataset, there are significant weaknesses with both these data and many other studies in the manuscript that diminish this enthusiasm considerably. I have detailed my specific comments for the authors to …
Reviewer #3 (Public Review):
This manuscript investigates the impact of Growth arrest-specific 6 (Gas6) on Zika virus (ZIKV) infection and congenital disease. The authors use a cohort of patient samples isolated from the recent ZIKV outbreak and compliment these data with in vitro and in vivo mouse models to conclude that Gas6 association with ZIKV viral particles promotes neuronal complication and congenital ZIKV syndrome. A clear strength of the study is the incorporation of human data, which allows for some translatability from the in vitro and in vivo mouse studies included in the study. However, despite enthusiasm for the human dataset, there are significant weaknesses with both these data and many other studies in the manuscript that diminish this enthusiasm considerably. I have detailed my specific comments for the authors to consider below:
General comments:
1. For data transparency, it would be nice for the authors to include individual data points throughout the manuscript. This is also important for patient data, where the authors only now show box plots and not individual data points.
2. The font size in many figures is very small and resolution is very low. The authors should consider increasing these.
Specific comments:
1. The data provided in Figure 1 with a large cohort of patient data is a strength of the study, although the magnitude of the changes in Gas6 are not particularly compelling. Perhaps the authors can provide more discussion on variation in patient titers, timing of infection, or other information that might explain these differences. The authors should also include sex distribution of the patient data.
2. I do not find the data in Figure 2 particularly compelling. Moreover, these data do not support the conclusion that Gas6 directly correlates with these changes. I suggest removing these data from the manuscript.
3. Some aspects of Figure 4 are difficult to reconcile. Why does warfarin reduce viral titers so significantly without impacting levels of vRNA?
4. ELISA or Luminex data should be included to compliment transcript data in Figure 4.
5. There are Gas6 KO mice commercially available that the authors could use to support their findings. Data from these animals would be a rigorous way to determine the direct impact of Gas6 on ZIKV infections.
6. How stable is the association between Gas6 and ZIKV particles used? How was this complexing and purification performed? How did the authors measure the efficiency of the association? There are very few experimental details provided on these very important points.
7. Additional animal numbers are needed for the data shown in Figure 5D and 5E unless the authors can provide some justification of the analyses used to calculate the power needed for these studies.
8. There are no details provided for the data in pregnant women. At what stage of gestation were these women? When was the collection of blood relative to timing of infection? These are important details that should be included.
9. There are insufficient n's to show the data in Supplemental Figure 6B.
10. Figure 6 is difficult to interpret given the overall low number of animals and lack of analyses. Given that each pup has its own placenta, how do the authors know which of the fetuses in which the dam was infected with Gas6-ZIKV were actually infected/exposed to the virus? The data in Figure 6F would suggest that every fetus was infected, which is difficult to imagine at this stage of gestation. More details should be provided.
11. The inclusion of IHC and H&E analyses from placentas is needed.
12. Were only two animals used to generate the data in Figure 6D? What is the justification for such low numbers?
13. The impact of the Gas6-ZIKV complex virus on maternal viral loads are a critical point and should be included (with appropriate n's).
14. The author's conclude that monocytes are the primary producers of Gas6, but do not provide sufficient data to support these claims. As these data were performed in THP-1-derived monocytes, the authors should consider differentiating these monocytes to macrophages using PMA and determine the impact of this modulation on Gas6 production.
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